The DNA tumor virus, SV40, has been an invaluable tool for studying molecular aspects of eukaryotic gene regulation, DNA replication, and viral oncogenesis. A major focus on the structure, function and regulated expression of the SV40 large T antigen has already revealed an unusually large number of important findings concerning viral and cellular processes. Thus far, our studies have been largely directed at understanding the structure and function of this interesting multi-functional regulatory protein. For example, a careful and systematic study of T antigen has provided insights into the mechanism of repression and autoregulation, initiation of viral DNA synthesis, viral coded enzymatic activities, sequence-specific protein-DNA interactions, and virally-induced oncogenesis. In this grant renewal, we turn our attention to a concerted and systematic effort at investigating how T antigen expression is regulated by the interplay of viral and cellular trans-activating factors. In addition, we will continue to use SV40 as a model to study complex regulatory processes occurring in the cells, such as transcription. At the same time, we hope to compare the mechanisms mediating expression of several other viral and cellular genes related to SV40 T antigen by the occurrence of common regulatory networks. A major effort will be devoted to identifying, purifying and characterizing various trans-activating cellular factors such as transcription factor Sp1, TATA binding factor (TBF), CAAT transcription factor (CTF), and enhancer binding protein (EBP), that are known to be involved with transcription in eukaryotic cells. Many of these transcription factors have been identified by biochemical fractionation and in vitro transcription studies of SV40, which revealed that they are DNA binding proteins. Thus, the methods that we have already developed for T antigen DNA binding experiments can now be applied to dissect the nature and specificity of the protein-DNA interactions for Sp1, TBF, CTF, and EBP. We will also raise antibodies directed against these proteins and attempt cloning of the genes encoding these factors. The antibodies and molecular clones will be used to help us unravel the mechanism of action and define potential protein- protein interactions that mediate transcriptional specificity. In addition, we plan to continue our studies of the SV40 large T antigen protein by devising large-scale isolation procedures, and attempting to crystallize and carry out X-ray diffraction studies of this virally-encoded protein. Finally, we will make use of in vitro DNA replication reactions to investigate the role of T antigen in triggering SV40-DNA replication and as a regulator of transcription.